INTRODUCTION

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Mammalian orthoreoviruses (reoviruses) provide useful models to study how viruses from the Reoviridae family in particular, and viruses lacking lipid envelopes in general, enter their host cells and initiate infection. Reovirus virions are 85-nm particles comprising the segmented double-stranded RNA genome enclosed by two concentric icosahedral protein capsids. The outer capsid mediates viral entry into the cytoplasm of host cells, where viral replication occurs. Outer capsid protein 1 (50 kDa, 36 copies) forms trimers that extend from the fivefold axes of virions and mediates viral attachment to cellular receptors. µ1 (76 kDa, 600 copies), found in virions mostly as fragments µ1N (4 kDa) and µ1C (72 kDa), participates in viral penetration of the cellular membrane barrier during entry. 3 (41 kDa, 600 copies), the major surface protein of virions, interacts closely with µ1, thereby controlling its conformational status. 2 (144 kDa, 60 copies) forms pentameric turrets that surround the fivefold axes and bridge the inner and outer capsids. 2 is involved in viral mRNA synthesis and assembly of the outer capsid onto virus particles but is not known to participate in entry. Several recent articles discuss the structure of reovirus virions and functions ascribed to the capsid proteins (30, 34, 48).

Reovirus entry into cells is a multistep process characterized by programmed disassembly of virions into at least two types of subvirion particles, each with specialized roles in infection (28, 34, 48). After binding to a receptor(s) at the cell surface, virions are taken up into the endocytic pathway. There, lysosomal proteinases act upon them to produce intermediates that resemble infectious subvirion particles (ISVPs) generated by in vitro proteinase treatment of virions (2, 14, 42). ISVPs lack 3, contain a cleaved form of µ1C, and may possess a conformer of 1 different from that in virions (9, 26, 29, 35, 40). These ISVP-like particles initiate penetration of cellular membranes, culminating in delivery of particles into the cytoplasm (10, 32, 42). Concomitantly with membrane penetration, virus particles are activated to synthesize the viral mRNAs. These transcriptase particles may resemble cores produced by in vitro proteinase treatment of virions or ISVPs (11, 28, 40). Cores lack µ1 and 1, contain a conformer of 2 different from that present in virions and ISVPs, and are transcriptionally active (11, 21, 29, 40).

The mechanisms by which outer capsid proteins 1, µ1, and 3 mediate the steps in viral entry remain to be fully elucidated. A major obstacle is the lack of a reverse genetics system to produce virions with mutations in these proteins. To mitigate this problem, we recently described a strategy termed "recoating genetics" that permits analysis of infectious particles containing engineered forms of µ1 and 3 (12, 27). Recoating genetics is enabled by the capacity of the recombinant proteins to bind and "recoat" purified subvirion particles in vitro, generating infectious particles that resemble virions: 3 binds ISVPs to produce recoated ISVPs, and µ1 and 3 bind cores to produce recoated cores (r-cores). However, recoated ISVPs and r-cores do not permit molecular genetic studies of the receptor-binding protein 1 because the former particles contain native 1 and the latter particles lack 1 altogether. In this report, we extend recoating genetics to the 1 protein by generating a new type of recoated cores that contains recombinant 1, µ1, and 3 (r-cores+1). These particles closely resemble native virions in structure, behavior in entry assays, and infectivity in cultured cells. Experiments with r-cores+1 demonstrated their utility for molecular genetic studies of 1 and provided new insights into the structure and assembly of reovirus particles.

	MATERIALS AND METHODS

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Cells and viruses. Spinner-adapted murine L929 fibroblast cells (L cells) (26), murine erythroleukemia cells (MEL cells) (39), Spodoptera frugiperda Sf21 insect cells (12), and Trichoplusia ni Tn High Five insect cells (Invitrogen) (12) were grown as described previously. Purified reovirus virions (26), ISVPs (26), and cores (12) were obtained as described previously. Particle concentrations were measured by A₂₆₀ (19, 41). Plaque assays to determine infectivities of reovirus preparations were performed as described previously (26). cDNA clones and recombinant baculoviruses for expressing µ1 and 3 together (12) or 1 alone (15, 22) have been described previously.

Expression of 1, µ1, and 3. Tn High Five cells were infected with fourth-passage virus stocks at a multiplicity of infection of 5 to 10 PFU/cell, and cells were harvested at 65 h postinfection. Cytoplasmic lysates of baculovirus-infected cells expressing µ1 and 3 only were prepared by lysis with Triton X-100 as described previously (12). Lysates containing 1, µ1, and 3 were generated as follows: cells were separately infected with 1- and µ1/3-expressing baculoviruses (1.5 × 10⁷ cells each), mixed together, resuspended in 1 ml of lysis buffer (100 mM NaCl, 1.5 mM MgCl₂, 250 mM sucrose, 10 mM Tris [pH 7.5]), lysed by probe sonication in an ice-ethanol bath (six 25-s pulses at 15 W), and centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was used to prepare r-cores+1. Cells were separately infected with the 1- and µ1/3-expressing baculoviruses because coinfection substantially reduced the yield of each protein at the multiplicity of infection used.

Preparation of r-cores, r-cores+1, and proteinase-treated r-cores+1. r-cores were prepared from insect cell lysates containing µ1 and 3 essentially as described previously (12). Briefly, lysates were incubated with purified T1L cores at a ratio of 6 × 10⁶ cell equivalents (400 µl of lysate) per 10¹² cores at 37°C for 2 h. The amounts of µ1 (200 µg) and 3 (100 µg) present in the lysate represented a twofold excess of protein relative to the amounts needed to fully recoat the cores. Reaction mixtures were chilled and then loaded atop 14-ml step gradients, each containing a preformed CsCl gradient ( = 1.25 to 1.55 g/cm³, 12 ml) and a 2-ml sucrose cushion (20% [wt/vol]). Gradients were centrifuged for 2 to 16 h in a Beckman SW-28 rotor at 25,000 rpm and 5°C. r-cores were recovered as an optically homogeneous band and were further purified by being loaded onto a preformed CsCl gradient ( = 1.25 to 1.45 g/cm³, 4 ml) and centrifuged overnight in a Beckman SW-50.1 rotor at 40,000 rpm and 5°C. The harvested particles were dialyzed extensively against virion buffer. r-cores+1 were prepared in the same way as were r-cores, except that lysates containing 1, µ1, and 3 were used at a ratio of 1.2 × 10⁷ cell equivalents (400 µl of lysate) per 10¹² cores. The amount of 1 (5 mg) present in the lysate represented a 500-fold excess of protein relative to the amount needed to fully recoat the cores. Concentrations of r-cores and r-cores+1 were measured by densitometry of Coomassie blue-stained gels as described previously (12). Proteinase-treated r-cores+1 were prepared from r-cores+1 by in vitro treatment with chymotrypsin as described previously (12).

Cryo-TEM and image reconstructions. Purified virions and r-cores+1 were embedded in vitreous ice, and micrographs were captured at a nominal magnification of 38,000× using low-dose transmission cryoelectron microscopy (cryo-TEM) procedures (4) on a Philips CM200FEG microscope. Micrographs were digitized at a 7-µm step size on a Zeiss PHODIS microdensitometer. Image pixels were bin averaged to obtain pixel sizes of 14 µm (3.68 Å) for virions and r-cores+1 and 21 µm (5.53 Å) for r-cores. The r-core data were obtained from previously captured micrographs (12), except that 194 instead of 228 particles were used and these were reprocessed to reduce the effects of the microscope contrast transfer function in the final density maps. The r-core+1 data consisted of 1,078 particles (eight micrographs; defocus values, 1.4- to 2.5-µm underfocus). The virion data consisted of 860 particles (five micrographs; defocus values, 1.4- to 2.8-µm underfocus). Reconstructions were computed using established icosahedral particle procedures (6, 25). Corrections to compensate in part for the effects of the microscope contrast transfer function were applied to image data in the reconstruction program with Wiener filtering as described previously (49). For all three particle types, orientation angles were evenly distributed throughout the asymmetric unit as shown by all inverse eigenvalues being <0.1 (25). Final density maps were calculated to a 24-Å resolution limit, deemed to be near or below the resolution of each of the data sets as measured by various R-factor, correlation coefficient, and phase difference calculations (5). For the r-core+1 minus r-core difference map (21, 47), the magnifications and density values of the maps were scaled for maximum correlation of densities between radii 220 and 450 Å. The scaled r-core map was then subtracted from the r-core+1 map and displayed at a density threshold approximately three times higher than that used for the other maps, thereby allowing visualization of the most significant portions of the difference map.

	RESULTS

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In vitro recoating of cores with recombinant 1, µ1, and 3 proteins. To test whether recombinant 1 can be added to cores in vitro along with µ1 and 3, a cell lysate containing all three proteins was incubated with purified cores, and virus particles were repurified by CsCl gradient centrifugation. Gel electrophoresis revealed that proteins comigrating with 1, µ1 and µ1C (henceforth termed µ1/µ1C), and 3 had bound to cores (Fig. 1). The identities of these proteins were confirmed by immunoblotting with polypeptide-specific antibodies (data not shown). No other proteins from the cell lysate were detected in the recoated particles. The cores recoated with 1, µ1/µ1C, and 3 (r-cores+1) in this study contained approximately stoichiometric amounts of µ1/µ1C and 3 relative to virions (data not shown), as previously shown for cores recoated with only µ1/µ1C and 3 (r-cores) (12). The amount of 1 in r-cores+1 varied between preparations but was always sufficient to be detected by Coomassie blue staining. In several preparations of r-cores+1, the amount of 1 approached that in virions (Fig. 1), suggesting that 1 can bind to cores in vitro with approximately native stoichiometry.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Protein composition of r-cores+1. Purified virions, cores, r-cores, and two preparations of r-cores+1 (no. 1 and no. 2) (8 × 1010 particles each) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) and Coomassie blue staining. Positions of viral proteins are indicated at left, and the position of 1 is highlighted with arrows.

r-cores+1 resemble virions in structure. Comparison of the image reconstruction of r-cores+1 with those of virions and r-cores revealed that all three types of particles are similar in overall appearance. Each possesses 600 finger-like projections at the particle surface attributed to the 600 subunits of 3, features beneath the 3 layer that represent the 600 subunits of µ1, and similar conformational states of the pentameric 2 turret (Fig. 2a to c). Image reconstructions of virions (Fig. 2a) and r-cores+1 (Fig. 2c) also contain an extended feature emerging from the center of the 2 turret that, in virions and ISVPs, has been attributed to 1 (21). The presence of this feature in r-cores+1 and its absence in r-cores (Fig. 2b) support the conclusion that r-cores+1, like virions, contain 1 bound at the fivefold axes.

View larger version (54K): [in this window] [in a new window]   FIG. 2.   Image reconstruction of r-cores+1. (a to c) Composite images comprising external (left half) and cutaway (right half) surface-shaded representations of virions (a), r-cores (b), and r-cores+1 (c) are shown. Features attributed to outer capsid proteins and the genome are labeled in panel a. (d) Difference map between r-cores+1 and r-cores. Only density features in the front half of the map are shown and are coded in red. (e) The difference map (red) superimposed upon a cutaway section of the r-core map. Features in the difference map at positions above and below the sectioning plane are not shown. (f) Magnified view of a portion of the image in panel e. One segment of the pentameric shutter that closes the 2 turret is labeled (*). Scale bar, 200 Å.

A feature attributable to 1 is present within the 2 turret. Difference maps between the r-core+1 and r-core reconstructions were computed to assess more carefully the effects of 1 assembly on particle structure (Fig. 2d). Only two significant density features, both located at the fivefold axes of r-cores+1, were seen in the difference map. The absence of significant differences in other locations confirmed that assembly of 1 into r-cores+1 causes no major rearrangements in the core proteins, µ1, 3, or regions of 2 distal from 1. Superposition of the two features upon a cutaway section of the r-core reconstruction (Fig. 2e and f) indicated that the upper feature represents the 1 fiber extending above 2 (see above). The lower feature is located in line with the upper feature, just beneath the pentameric "shutter" that closes the top of the 2 turret. This feature might represent either a portion of 1 or fivefold-proximal 2 sequences that have undergone rearrangement upon 1 binding. However, we consider the latter unlikely because all of the density associated with 2 in r-cores is also present at similar positions in r-cores+1 (i.e., no significant loss of density from the 2 region was seen in the inverse difference map of the r-core and r-core+1 reconstructions [data not shown]). Therefore, we conclude that this lower feature represents the base of the 1 fiber that protrudes through the 2 shutter and into the turret cavity.

r-cores+1 bind efficiently to RBCs and L cells. To assess the competence of recombinant 1 in r-cores+1 for binding to cell receptors, we measured the capacity of virions, cores, r-cores, and r-cores+1 to induce hemagglutination (HA) of human erythrocytes (RBCs) (Fig. 3a). 1 mediates HA via its interactions with one or more glycoprotein on the cell surface (1, 38). Virions induced HA efficiently as expected, while cores and r-cores, which lack 1, failed to do so even at the highest particle concentration tested (fivefold higher than the maximum shown in Fig. 3a [data not shown]). r-cores+1 agglutinated RBCs nearly as efficiently as did virions, indicating that these particles containing recombinant 1 are functional for attachment to the reovirus receptor(s) on human RBCs.

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Capacity of r-cores+1 to bind to cells. (a) HA induced by purified virions, cores, r-cores, and r-cores+1 was determined by endpoint titration using human type A+ RBCs (American Red Cross), as described previously (20). Serial twofold virus dilutions were incubated with RBCs (0.4% [vol/vol]) in a 96-well round-bottomed plate at 4°C for 2 h, and HA was visually detected. The number of HA units induced by a virus preparation is given by the highest number of particles used (2 × 1010 particles) divided by the minimum number of particles required to induce HA. HA activity is expressed as the log2(HA units). Averages ± standard deviations of three replicates are shown. (b) Attachment of purified virions, r-cores, and r-cores+1 to L cells at 4°C was determined by indirect immunofluorescence as described previously (18). Cells on coverslips were incubated with purified virus (5 × 105 particles/cell) at 4°C for 1 h. Unbound virus was removed by washing with ice-cold phosphate-buffered saline. After the cells were fixed and permeabilized, bound virus was detected by using a 1:500 dilution of rabbit antireovirus serum (44) as primary antibody and a 1:100 dilution of fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G (Pierce) as secondary antibody.

r-cores+1 are nearly as infectious as virions and display similar growth kinetics. r-cores containing levels of µ1/µ1C and 3 similar to those of virions but lacking 1 were 250 to 500 times more infectious in L cells than were precursor cores but only ~0.0001 times as infectious as virions on a per-particle basis (12) (Fig. 4a). r-cores+1 containing levels of 1 similar to those of virions (Fig. 1) were 3,000 times more infectious than r-cores and 0.5 times as infectious as virions on a per-particle basis (Fig. 4a), consistent with the capacity of r-cores+1 and virions, but not r-cores, to attach to cells efficiently (Fig. 3b). Infection by virions and r-cores+1, but not r-cores, was blocked by anti-1 antibody 5C6 (Fig. 4b) (45), showing that the enhanced infectivity of r-cores+1 compared with that of r-cores is due to recombinant 1 in the former particles.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Infectious properties of r-cores+1 in L cells. (a) The infectivities of purified virions, cores, r-cores, and r-cores+1 were determined by plaque assay on L-cell monolayers and expressed as PFU per milliliter (26). After standardization against the number of virus particles per milliliter, the relative infectivity of each preparation was determined by dividing its PFU per milliliter by the PFU of cores per milliliter. Averages of three replicates are shown (standard deviation  0.10 log10 units). (b) The capacity of anti-T1L 1 antibody 5C6 to neutralize formation of plaques on L cells by purified virions, r-cores, and r-cores+1 was determined as described previously (12). Virus (100 PFU) was incubated with 5C6 (1 µg/ml) for 1 h at 37°C, and infectivity was measured by plaque assay. The extent of neutralization for each preparation (expressed as percent plaque survival) was the ratio of PFU obtained in the presence of antibody to the PFU obtained in its absence. Averages ± standard deviations of three replicates are shown. (c) Single-cycle growth curves of purified virions, r-cores+1, and chymotrypsin treatment mixtures containing ISVPs and proteinase-treated r-cores+1 (pr-core+1) (2 PFU/cell) were generated in L cells as described previously (13). Infectivity at a specified time (t) relative to time zero was expressed as log10(PFU/ml)t  log10(PFU/ml)0. Each data point represents the average of duplicate values.

r-cores+1 recapitulate strain-dependent differences attributed to 1. Analysis of r-cores+1 assembled with different types of recombinant 1 provides a new approach for genetic studies of particle-bound forms of this protein. To evaluate the feasibility of this approach, we produced r-core+1L and r-core+1D particles containing genome and core, µ1/µ1C, and 3 proteins from reovirus strain type 1 Lang (T1L) but recombinant 1 from strain T1L or type 3 Dearing (T3D), respectively. We then determined the properties of r-cores+1L and r-cores+1D in the following assays that show a 1-dependent difference between native T1L and T3D virions.

(i) HA of bovine RBCs (Fig. 5a). T3D virions induce HA of bovine RBCs via interactions between T3D 1 and RBC sialoglycoproteins (20, 38). T1L virions do not agglutinate bovine RBCs, likely because T1L 1 does not bind sialic acid and no other receptors are available for it on these cells (20, 38). r-cores+1D induced levels of HA similar to those induced by T3D virions, while r-cores+1L, like T1L virions, failed to induce HA. Thus, the capacity of r-cores+1 to agglutinate bovine RBCs is determined by the origin of the particle-bound 1 protein, consistent with previous genetic and biochemical evidence (20, 38).

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Capacity of r-cores+1 to mediate 1-specific in vitro and in vivo properties. (a) HA of bovine RBCs (Colorado Serum Co.) induced by purified T1L virions, T3D virions, r-cores+1L, and r-cores+1D was determined as described for Fig. 3a. Averages ± standard deviations of three replicates are shown. (b) The effect of chymotrypsin treatment on infectivity of T1L virions (open squares), T3D virions (open circles), r-cores+1L (filled squares), r-cores+1D (filled circles), and r-cores+1D(T249I) (filled triangles) was determined as follows. Purified virus (1.5 × 1012 particles/ml) was incubated with chymotrypsin (200 µg/ml) at 37°C. At specified times, an aliquot was removed onto ice and digestion was stopped with phenylmethylsulfonyl fluoride (2 mM). The infectivity of each aliquot was determined by plaque assay. Viral infectivity at a specified time (t) relative to time zero was expressed as described for Fig. 4c. Averages ± standard deviations of three independent experiments are shown. (c) Infectious yields produced in MEL and L cells by T1L virions, T3D virions, r-cores+1L, and r-cores+1D were determined as follows. Purified virus (5 PFU/cell) was incubated with 4 × 106 cells for 1 h at room temperature. Unbound virus was removed by centrifugation (500 × g) and washing cells with phosphate-buffered saline. Cells were then resuspended in growth medium and incubated at 37°C for 24 h. Cell lysates were generated by freeze-thawing, and the amount of infectious virus in these lysates was determined by plaque assay. Growth yields were expressed as log10(PFU/ml) at 24 h. Averages ± standard deviations of six trials from two independent experiments are shown.

(ii) Effect of in vitro chymotrypsin treatment on viral infectivity (Fig. 5b). When T1L virions are treated with chymotrypsin in vitro, infectivity increases at early times and is then maintained for at least 2 h. In contrast, the infectivity of T3D virions increases transiently upon chymotrypsin treatment but then decreases to plateau at 10% of the starting value (36). r-cores+1L and r-cores+1D behaved like T1L and T3D virions, respectively, upon chymotrypsin treatment, confirming the previous hypothesis (15, 36) that these effects of chymotrypsin on infectivity are determined by the origin of particle-bound 1.

(iii) Viral growth in MEL cells (Fig. 5c). T3D virions produce ~10,000 times higher yields of infectious progeny than do T1L virions in MEL cells, whereas the two types produce similar yields in L cells (16, 39). This strain-dependent difference in viral growth has been genetically mapped to the S1 gene segment, which encodes the 1 and 1s proteins (39). Moreover, viral strains adapted to grow in MEL cells contain mutations in 1 that increase their capacity to bind to MEL cells (16). Findings to date therefore support a model in which the capacity of reoviruses to infect and grow in MEL cells is determined by 1-receptor interactions. It remained possible, nevertheless, that the above difference in viral growth arises not from 1 protein present in infecting particles but from 1 transcripts or 1 or 1s polypeptides newly synthesized in infected cells. To test that possibility, we measured the growth yields of r-cores+1L and r-cores+1D in MEL and L cells and found that these mirrored the yields obtained with native T1L and T3D virions, respectively. Because r-cores+1L and r-cores+1D possessed the same genome (T1L) and differed only in the origin of their particle-bound 1 proteins, our results confirm that the growth difference between T1L and T3D viruses in MEL cells arises from differences in viral entry attributable to particle-bound 1, likely at the cell attachment step.

	DISCUSSION

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The 1-2 interaction in reovirus particles. Protein 1 forms lollipop-shaped homotrimers consisting of a base, a long fibrous tail, a neck, and a globular head (7, 15, 24, 26, 37). The amino-terminal base of 1 anchors it to the particle (26, 31), while more carboxy-terminal sequences in the neck and head domains mediate binding to cellular receptors (16, 20, 23, 33). Image reconstructions of virions and ISVPs show that the base of each 1 fiber is anchored to the particle by interaction with the pentameric shutter atop each 2 turret (21). However, little is known about the structural details of the 1-2 binding interaction because density features contributed to the binding interface by each protein cannot be distinguished in the cryo-TEM maps of native particles. Comparison of the r-core and r-core+1 image reconstructions in this report suggests that the base of the 1 fiber protrudes through the center of the pentameric 2 shutter and that these protruding 1 sequences form a small knob-like domain within the large cavity enclosed by the 2 turret. On the basis of this observation, we speculate that the 1 fiber remains anchored to 2 in virions and ISVPs at least in part because its internal knob domain cannot escape through the narrow channel at the center of the 2 shutter in those particles (12, 21). Consistent with that idea, 1 binds poorly to cores (K. Chandran and M. L. Nibert, unpublished data), which contain a larger channel at the center of each open 2 turret (21).

The region(s) of 1 visualized in the cryo-TEM image reconstruction of r-cores+1. Electron microscopic studies of negatively stained preparations of purified 1 fibers indicate that each is 480 to 500 Å in length, with the N-terminal tail and C-terminal head portions comprising 380 to 400 and 100 Å of the fiber, respectively (24, 26). The density features attributed to 1 in the r-core+1 reconstruction, however, measure only about 140 Å in length. If the 2-proximal portion of the 1 tail assumes an extended conformation after assembly into r-cores+1, then the density visualized in the r-core+1 reconstruction represents only the first 100 to 120 residues of 1 according to the current model for sequence-structure correlations in the fiber (24). This region of the 1 tail comprises the putative N-terminal 2-binding domain and approximately one-half of the long -helical coiled coil (24, 30, 31, 37). More distal tail and head sequences are not visualized in the cryo-TEM map, probably because those regions of the 1 fiber are more variably placed relative to the icosahedral lattice, due to fiber bending. This idea is supported by calculations for position-dependent flexibility of the 1 fiber from electron micrographs as well as from the amino acid sequence: both analyses found a local maximum in fiber flexibility at a distance of about 140 Å along the tail (24). r-cores+1 containing "stiffened" forms of recombinant 1 may allow visualization of more distal domains in the fiber.

Infectious properties of r-cores+1. Assembly of approximately stoichiometric amounts of recombinant 1, µ1, and 3 onto cores to produce r-cores+1 boosted the infectivity of cores by nearly a millionfold, to ~50% of that of virions. This almost complete reconstitution of viral infectivity suggests that r-cores+1 are free from major defects. Moreover, observations that r-cores+1 and proteinase-treated r-cores+1 replicated in L cells with kinetics identical to those of virions and ISVPs, respectively, and that r-cores+1, like virions, required acid-dependent lysosomal proteinase(s) for infection strongly suggest that these recoated particles utilize the same pathways as do native particles for productive entry. Hence, r-cores+1 are valid and powerful tools for entry studies.

Utility of r-cores+1 for genetic studies of 1. Experiments performed with a variety of reovirus strains and cultured cell types indicate that 1 is the primary determinant of cell tropism. The 1-encoding S1 gene segment is also responsible for a number of strain-dependent patterns of viral pathogenesis in infant mice, including the capacity of viruses to infect and replicate in intestinal tissue (see below) and to replicate in neurons in the central nervous system (46). S1 is also a genetic determinant of strain-specific differences in the capacities of reoviruses to induce apoptosis in cultured cells (43). Most, if not all, of these phenotypic differences mapping to S1 may relate to the role of 1 as a receptor-binding protein, suggesting that they should be amenable to analysis with r-cores+1. In the current study, infections with r-cores+1 containing T3D or T1L 1 proved that the tropism of T3D, but not T1L, virions for MEL cells is determined by particle-bound 1. Further analysis of this and other phenotypic differences between T1L and T3D viruses by using r-cores+1 that contain chimeric forms of 1 (17) should aid in dissecting functional domains of this protein.